Gluconeogenesis is the metabolic pathway for the biosynthesis of glucose from non-carbohydrate precursors including pyruvate, lactate, and citric acid cycle intermedates. This pathway occurs predominantly in the liver and to a lesser extent in the kidney and is triggered by a fall in blood glucose concentration. Gluconeogenesis meets the needs of the body for glucose when there is insufficient intake of carbohydrates in the diet. It is also critical to the maintenance of a continuous energy supply, in the form of glucose, to red blood cells and tissues of the central nervous system, which do not undergo gluconeogenesis. In addition, gluconeogenesis represents a mechanism by which products of metabolism from other tissues are cleared from the blood and converted back into glucose. Under fasting conditions glucose production through this pathway maintains the basal glucose concentrations necessary to sustain primary physiologic functions. The gluconeogenic pathway is essentially glycolysis (the breakdown of glucose received predominantly from the diet) in reverse. There are four enzymes necessary to bypass the thermodynamically unfavorable steps of glycolysis. These enzymes are pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase. The rate-limiting step of gluconeogenesis is catalyzed by phosphoenolpyruvate carboxykinase and this enzyme has been well characterized in several species. Two forms (isozymes) of the enzyme have been isolated and the total enzyme activity displayed in humans is equally divided between the cytosolic and mitochondrial forms (Hanson and Patel, Adv. Enzymol. Relat. Areas Mol. Biol., 1994, 69, 203-281).
Cytosolic phosphoenolpyruvate carboxykinase (also known as PCK1, cyPCK and PEPCK-C) is expressed predominantly in liver where it acts in the gluconeogenic pathway and in kidney where it acts in the gluconeogenic pathway as well as being glyceroneogenic and ammoniagenic (Hanson and Reshef, Annu. Rev. Biochem., 1997, 66, 581-611). PEPCK-cytosolic also exhibits significant expression in white and brown adipose tissue, lactating mammary gland and the small intestine where it is thought to supply glycerol for triglyceride synthesis (glyceroneogenesis) in these tissues (Hanson and Reshef, Annu. Rev. Biochem., 1997, 66, 581-611).
Studies using transgenic mice have shown that different cis acting elements are required to drive the expression of PEPCK-cytosolic in hepatocytes, renal tubule epithelial cells and adipocytes (Beale et al., Faseb J., 1992, 6, 3330-3337).
The overall expression of PEPCK-cytosolic is controlled entirely at the level of transcription by a wide variety of physiological stimuli including dietary carbohydrate, hormones, and cellular intermediates (Hanson and Patel, Adv. Enzymol. Relat. Areas Mol. Biol., 1994, 69, 203-281). It is expressed in the periportal region of the liver and is therefore sensitive to oxygen concentration. Studies of rat hepatocytes demonstrated that the glucagon-dependent activation of the PEPCK-cytosolic gene is modulated by oxygen and that this process is mediated by hydrogen peroxide (Kietzmann et al., Kidney Int., 1997, 51, 542-547; Kietzmann et al., FEBS Lett., 1996, 388, 228-232). Other factors have also been shown to impair the glucagon-induced increase in PEPCK-cytosolic including interleukin-1-beta, tumor necrosis factor alpha and interleukin-6 (Christ and Nath, Biochem. J., 1996, 320, 161-166; Christ et al., Hepatology, 1997, 26, 73-80).
In the liver, PEPCK-cytosolic is negatively regulated by insulin and has therefore been considered a potential contributing factor to hyperglycemia in diabetics (Sutherland et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci., 1996, 351, 191-199). Studies using various kinase inhibitors demonstrated a link between PEPCK-cytosolic gene regulation by insulin and the protein kinase phosphatidylinositol-3-kinase (PI3-kinase). In these studies, it was shown that insulin inhibition of PEPCK-cytosolic gene expression requires PI3-kinase but that the signal is not mediated by MAP kinases nor transmitted through protein kinase B or protein kinase C (Agati et al., J. Biol. Chem., 1998, 273, 18751-18759; Sutherland et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci., 1996, 351, 191-199).
PEPCK-cytosolic gene expression is also sensitive to other regulatory stimuli including cell volume. It has been shown that in rat and human hepatocytes, changes in cell volume alter the rate of transcription and mRNA stability of PEPCK-cytosolic (Kaiser, Am. J. Physiol., 1998, 274, G509-517).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of PEPCK-cytosolic and to date, strategies aimed at investigating PEPCK-cytosolic function have involved the use of chemical inhibitors and transgenic mice.
Consequently, there remains a long felt need for agents capable of effectively inhibiting PEPCK-cytosolic function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of PEPCK-cytosolic expression.